Known SNP genotyping.

This is one of the highly studied methods in the biology field. After the invention of PCR by Mullis a dynamic changes has occurred in this field. Broadly we can classify this in to 2 ,one is conventional PCR RFLP and the other one is multiplexing.

PCR RFLP: In this we will amplify a short segment of DNA which contains our SNP and will cut the amplified DNA with help of an enzyme. These enzymes are known as restriction enzymes. Restriction enzymes are nothing but DNA cutters which cuts DNA at a particular sequence for example EcoR I enzymes the DNA which is having the sequence G/AATTC (/ indicates the site of cut). In this case if we want to see whether GAATTC the A in red is mutated or not. We can need to amplify 100-200 basepairs DNA around this particular sequence and the after amplification we have to incubate the amplicon along with the enzyme. After that we need to run the digested product on to a gel and then we have to see whether the enzyme cut the DNA or not. If the enzyme cuts the DNA means there is no mutation at that particular base and the restriction site is preserved. If there is any mutation the enzyme will not cut the amplified DNA. This method is old fashioned today because it consumes much time when compared to the other applications available. Another restriction is we may not get the enzymes for all the sites which we want to screen.

TaqMan. This application is very rapid and accurate. This method works on the principle that the Taq polymerase have 5’ exonuclease activity. In this we will have a primerset and a probe which is labelled with a Vic and florescent Dye. A Vic blocks the Dyes florescent activity until it is with the probe. If the probe get digested the Vic and Dye will be separated then florescence of the dye will be detected by the system.

In SNP detection we will design a probe in such a condition which will be specific for our SNP and a primer set with the specificity of our (ROI)
this will be further explained in future posts be in touch

Methods to detect SNPs

There are several methods to SNP identification.
SNP identification can be categorized in to 2 main sub sets.
1. Novel SNP detection.
2. Simply detecting the known SNPs.

For novel snp detection sequencing will be the best method. In this we will select the region which we want to screen for snps from a public database like Ensembl and/or NCBI and we will sequence the region using primers which are specific for that particular region. Once we sequence the region of interest (ROI) we have to compare the sequence to the preexisting sequence to see the variations(SNPs). Here we will get a doubt if we don't have a previous sequence knowledge of that ROI the how can we identify the SNPs. In such case if you are the first person to perform the sequence analysis for that ROI then you have to do the same thing with some other samples after that compare all the sequence to each other and then you will come to know whether there is any variation present in that ROI. To do this we have to use sequence alignment softwares. These softwares align sequences to each others, by doing this we can compare them very fast and very easily. Once we get an idea about the SNp sites in our ROI the we can go for known SNP detection.
Known SNP detection is also known as SNP genotyping. There are several methods available for this. We will discuss about them in future.

If you have any observations please don't hesitate to contact us. Be in touch........

Single Nucleotide Polymorphism(SNP)

A single nucleotide polymorphism, or SNP (pronounced snip), is a DNA sequence variation occurring when a single nucleotide - A, T, C, or G - in the genome (or other shared sequence) differs between members of a species (or between paired chromosomes in an individual). For example, two sequenced DNA fragments from different individuals, AAGCCTA to AAGCTTA, contain a difference in a single nucleotide. In this case we say that there are two alleles : C and T. Almost all common SNPs have only two alleles.

Within a population, SNPs can be assigned a minor allele frequency - the ratio of chromosomes in the population carrying the less common variant to those with the more common variant. It is important to note that there are variations between human populations, so a SNP allele that is common in one geographical or ethnic group may be much rarer in another. In the past, single nucleotide polymorphisms with a minor allele frequency of greater than or equal to 1% (or 0.5%, etc.) were given the title "SNP," an unwieldy definition. With the advent of modern bioinformatics and a better understanding of evolution, this definition is no longer necessary.

DNA strand 1 differs from DNA strand 2 at a single base-pair location (a C/T polymorphism).

Single nucleotide polymorphisms may fall within coding sequences of genes, non-coding regions of genes, or in the intergenic regions between genes. SNPs within a coding sequence will not necessarily change the amino acid sequence of the protein that is produced, due to degeneracy of the genetic code. A SNP in which both forms lead to the same polypeptide sequence is termed synonymous (sometimes called a silent mutation) - if a different polypeptide sequence is produced they are non-synonymous. SNPs that are not in protein-coding regions may still have consequences for gene splicing, transcription factor binding, or the sequence of non-coding RNA.

Variations in the DNA sequences of humans can affect how humans develop diseases and respond to pathogens, chemicals, drugs, vaccines, and other agents. However, their greatest importance in biomedical research is for comparing regions of the genome between cohorts (such as with matched cohorts with and without a disease).

The study of single nucleotide polymorphisms is also important in crop and livestock breeding programs (see genotyping). See SNP genotyping for details on the various methods used to identify SNPs.

hematopoietic stem cells

fig: Hematopoietic and Stromal Stem Cell Differentiation.

With more than 50 years of experience studying blood-forming stem cells called hematopoietic stem cells, scientists have developed sufficient understanding to actually use them as a therapy. Currently, no other type of stem cell, adult, fetal or embryonic, has attained such status. Hematopoietic stem cell transplants are now routinely used to treat patients with cancers and other disorders of the blood and immune systems. Recently, researchers have observed in animal studies that hematopoietic stem cells appear to be able to form other kinds of cells, such as muscle, blood vessels, and bone. If this can be applied to human cells, it may eventually be possible to use hematopoietic stem cells to replace a wider array of cells and tissues than once thought.

Scientists face major roadblocks in expanding their use beyond the replacement of blood and immune cells. First, hematopoietic stem cells are unable to proliferate (replicate themselves) and differentiate (become specialized to other cell types) in vitro (in the test tube or culture dish). Second, scientists do not yet have an accurate method to distinguish stem cells from other cells recovered from the blood or bone marrow. Until scientists overcome these technical barriers, they believe it is unlikely that hematopoietic stem cells will be applied as cell replacement therapy in diseases such as diabetes, Parkinson's Disease, spinal cord injury, and many others.

Blood cells are responsible for constant maintenance and immune protection of every cell type of the body. This relentless and brutal work requires that blood cells, along with skin cells, have the greatest powers of self-renewal of any adult tissue.

The stem cells that form blood and immune cells are known as hematopoietic stem cells.They are ultimately responsible for the constant renewal of blood—the production of billions of new blood cells each day. Physicians and basic researchers have known and capitalized on this fact for more than 50 years in treating many diseases. The first evidence and definition of blood-forming stem cells came from studies of people exposed to lethal doses of radiation in 1945.

Basic research soon followed. After duplicating radiation sickness in mice, scientists found they could rescue the mice from death with bone marrow transplants from healthy donor animals. In the early 1960s, Till and McCulloch began analyzing the bone marrow to find out which components were responsible for regenerating blood .They defined what remain the two hallmarks of an HSC: it can renew itself and it can produce cells that give rise to all the different types of blood cells .

A hematopoietic stem cell is a cell isolated from the blood or bone marrow that can renew itself, can differentiate to a variety of specialized cells, can mobilize out of the bone marrow into circulating blood, and can undergo programmed cell death, called apoptosis—a process by which cells that are detrimental or unneeded self-destruct.

A major thrust of basic HSC research since the 1960s has been identifying and characterizing these stem cells. Because HSCs look and behave in culture like ordinary white blood cells, this has been a difficult challenge and this makes them difficult to identify by morphology (size and shape). Even today, scientists must rely on cell surface proteins, which serve, only roughly, as markers of white blood cells.

Identifying and characterizing properties of HSCs began with studies in mice, which laid the groundwork for human studies. The challenge is formidable as about 1 in every 10,000 to 15,000 bone marrow cells is thought to be a stem cell. In the blood stream the proportion falls to 1 in 100,000 blood cells. To this end, scientists began to develop tests for proving the self-renewal and the plasticity of HSCs.

The "gold standard" for proving that a cell derived from mouse bone marrow is indeed an HSC is still based on the same proof described above and used in mice many years ago. That is, the cells are injected into a mouse that has received a dose of irradiation sufficient to kill its own blood-producing cells. If the mouse recovers and all types of blood cells reappear (bearing a genetic marker from the donor animal), the transplanted cells are deemed to have included stem cells.

These studies have revealed that there appear to be two kinds of HSCs. If bone marrow cells from the transplanted mouse can, in turn, be transplanted to another lethally irradiated mouse and restore its hematopoietic system over some months, they are considered to be long-term stem cells that are capable of self-renewal. Other cells from bone marrow can immediately regenerate all the different types of blood cells, but under normal circumstances cannot renew themselves over the long term, and these are referred to as short-term progenitor or precursor cells. Progenitor or precursor cells are relatively immature cells that are precursors to a fully differentiated cell of the same tissue type. They are capable of proliferating, but they have a limited capacity to differentiate into more than one cell type as HSCs do. For example, a blood progenitor cell may only be able to make a red blood cell

Organization and diversity of cells

All organisms more complex than viruses consist of cells. All cells are derived by cell division from other cells. Ultimately, there must be an unbroken chain of cells leading back to the first successful primordial cell that lived maybe 3.5 billion years ago. How that cell formed is an interesting question.

Prokaryotes lack a defined nucleus and internal organisation is simple. Under the electron microscope they appear featureless. They comprise two kingdoms of life: eubacteria which include most of the bacteria; and the archaea, resemble bacteria and often grow in unusual environments, such as in acid hot springs, saturated brines, etc. The genome of a prokaryote typically consists of a single small circular chromosome in which the DNA is not packaged in any obviously organized way. Prokaryotes may be simple, but they are not primitive .

Fig: Prokaryotic and eukaryotic cell anatomy.

Eukaryotes are thought to have first appeared about 1.5 billion years ago.The organization of the Eukaryotes is complex. Membrane bound- organelles including nucleus are present. Eukaryotic cells have several linear chromosomes in their cell nuclei, in each of which a single very long DNA molecule is elaborately packaged by histone and other proteins. The number and DNA content vary greatly between species .In general the genome size tends to parallel the complexity of the organism, but there are many exceptions. Humans do not have especially large genomes, while the cells of an onion and a lily contain respectively about five and 30 times as much DNA as a typical human cell.

DNA Extraction from Plasma and Serum

1. Introduction

There are occasions where the only materiel available on a patient is stored plasma or

serum samples. In normal individuals, the amount of DNA in these samples is very low

but sufficient to serve as template for PCRs. Moreover, increased amounts of circulating

DNA have been found in a variety of disorders, including cancer, autoimmune disease,

and infection. Additionally, small amounts of fetal DNA have been detected in maternal

plasma/serum during gestation. We have used the following protocol to successfully

genotype archival plasma samples.

2. Materials

1. 10X SDS /Protein K: (Lauryl sulphate [SDS] 10 g/100 mL, Proteinase K 5 mg/mL).

2. TE (Tris EDTA) buffer: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0.

3. Phenol��chloroform (1��1 v/v).

4. Glycogen (10 mg/mL).

5. 7.5 M Ammonium acetate.

6. 100% ethanol.

7. 70% ethanol.

3. Method

1. Place 1.5 mL of serum or plasma into a 15-mL centrifuge tube.

2. Add 1.5 mL of 1X SDS proteinase K solution in the tube containing the serum and mix

well.

3. Digest overnight at 55°C in water bath.

4. Add 3 mL of phenol/chloroform solution.

5. Vortex 30 s and centrifuge for 10 min at 1000g using a swing-out rotor.

6. Transfer aqueous layer to fresh tube and repeat steps 4 and 5.

7. Transfer aqueous layer to fresh tube and add 5 ìL of glycogen (10 mg/L), 1 mL of 7.5 M

ammonium acetate, and 8 mL of 100% ethanol.

8. Mix by inverting and centrifuge at 2500g for 40 min.

9. Carefully remove supernatant and wash pellet in 10 mL of 70% ethanol.

10. Centrifuge at 2500g for 10 min. Carefully remove last traces of ethanol, and allow to air

dry for 10 min before redissolving in 100 ìL of TE.

RNA Extraction from Blood

1. Introduction

Based on the method of Chomczynski and Sacchi (1), this is an extremely reliable

method without the requirement for centrifugation over CsCl gradients. As with any

RNA protocol, extreme care should be taken to exclude RNAse contamination, the

greatest source of which will be the sample itself. All disposables and reagents should

be RNAse free.

2. Materials

1. Microfuge tubes (1.5 mL).

2. Ice bucket.

3. Microfuge.

4. Red cell lysis buffer: 1.6 M sucrose, 5% Triton X-100, 25 mM MgCl2, 60 mM Tris-HCl,

pH 7.5; stored at 2–8°C and used cold.

5. Extraction Buffer: 5.25 M guanidinium thiocyanate, 50 mM Tris-Cl, pH. 6.4, 20 mM

EDTA, 1% Triton X-100, 0.1 M â-mercaptoethanol (add immediately prior to use).

6. 2 M sodium acetate, pH 4.0.

7. Phenol (saturated with 1 M Tris-HCl: 0.1 M EDTA, pH 8.0).

8. Chloroform:Iso-amyl alcohol (24��1).

9. Isopropyl alcohol.

10. 70% Ethanol.

11. RNAse-free distilled water.

3. Method

1. In a microfuge tube, mix 100 ìL anticoagulated blood with 1 mL of red cell lysis buffer

(see Notes 1–3).

2. Leave at room temperature with occasional shaking until the red cells have lysed and the

solution translucent (usually within 5 min).

3. Microfuge for 30 s at 13,000g to pellet the white blood cells. Remove and discard

supernatant.

4. Add 200 ìL of extraction buffer and resuspend cell pellet by drawing through narrow

gauge needle several times.

5. Add 20 ìL of 2 M sodium acetate and mix gently by inversion.

6. Add 220 ìL of phenol and mix gently by inversion.

7. Add 60 ìL of chloroform/isoamyl alcohol (24��1) and vortex vigorously.

8. Place on ice for 15 min.

9. Microfuge at 12,000g for 5 min and transfer the upper phase to new microfuge tube.

10. Add 200 ìL of ice-cold isopropanol mix and store at –20°C for 30 min.

11. Microfuge at 12,000g for 15 min and discard supernatant.

12. Resuspend pellet in 200 ìL of extraction buffer.

13. Repeat steps 3 through 9.

14. Wash pellet with 400 ìL of cold 70% ethanol.

15. Microfuge at 12,000g for 5 min and discard supernatant.

16. Carefully remove last traces of ethanol from tube (folded sterile swab or kimwipe works

well).

17. Resuspend in 100 ìL of distilled water and incubate at 50°C for 15 min to dissolve RNA

(see Note 4).

4. Notes

1. Blood stored at room temperature or 4°C should be mixed thoroughly prior to aliquots

being removed.

2. Frozen blood samples should be allowed to thaw completely and mixed thoroughly before

aliquots being removed. Although freezing lyses red blood cells, the red cell lysis step

should still be performed to efficiently remove hemoglobin from the sample. Repeated

freeze/thaw cycles should be avoided.

3. Buffy coat contains two to four times the amount of white blood cells per volume compared

to fresh blood. Therefore, it is advisable to use only 50 ìL of buffy coat diluted with 50 ìL

of phosphate-buffered saline as starting material for this protocol.

4. Repeat pipetting through a narrow gauge tip can help this process.